Lead(II)-Binding Properties of the 5'-Monophosphates of Adenosine

potentiometric pH titrations in aqueous solution (25 °C; I ) 0.1 M, NaNO3). Based on previously ..... and absence of 0.93 mM Guo under N2 with 3 mL o...
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Inorg. Chem. 2000, 39, 5985-5993

5985

Lead(II)-Binding Properties of the 5′-Monophosphates of Adenosine (AMP2-), Inosine (IMP2-), and Guanosine (GMP2-) in Aqueous Solution. Evidence for Nucleobase-Lead(II) Interactions Carla P. Da Costa and Helmut Sigel* Institute of Inorganic Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel, Switzerland ReceiVed June 29, 2000

The stability constants of the 1:1 complexes formed between Pb2+ and the nucleosides (Ns), adenosine and guanosine, as well as between the nucleotides (NMP2-), AMP2-, IMP2-, and GMP2-, were determined by potentiometric pH titrations in aqueous solution (25 °C; I ) 0.1 M, NaNO3). Based on previously established log Pb H KPb(R-PO versus pKH(R-PO straight-line plots (R-PO32- ) simple phosphate monoester or phosphonate ligands 3) 3) where R is a noninteracting site), it is shown that the Pb(IMP) and Pb(GMP) complexes are more stable than is expected on the basis of the basicity of the phosphate group of IMP2- and GMP2-. This means that macrochelates are formed, where the phosphate-coordinated Pb2+ also interacts with N7 of the nucleobase residue. In contrast, the stability of the Pb(AMP) complex is governed by the basicity of the AMP2- phosphate group. These results agree with the observations made for the Pb(Ns)2+ complexes: Pb(adenosine)2+ is very unstable in contrast to Pb(guanosine)2+, the stability of which is very similar to the one of Pb(cytidine)2+ studied previously. The stability constants of the Pb(Ns)2+ complexes also allowed an evaluation of the structure in solution of the monoprotonated Pb(H;NMP)+ complexes, the stabilities of which were also determined. We were able to show that the proton is located at the phosphate group and Pb2+ at the N7/(C6)O site of H(GMP)-; in the case of H(AMP)- Pb2+ is probably about equally distributed between the adenine residue and the monoprotonated phosphate group. On the basis of the stability constants of these complexes and their structures in solution, it is possible to provide a series which reflects the decreasing affinity for Pb2+ of nucleobase residues in single-stranded nucleic acids: guanine = cytosine > (hypoxanthine) > adenine > uracil = thymine. The Pb2+ affinity of the phosphodiester linkage, -PO3--, is similar to the one of the adenine residue, but is expected to be more significant due to its larger abundance. The relevance of these results for lead-activated ribozymes is briefly discussed.

1. Introduction Lead has been recognized as a toxic element for many centuries.1 It produces a variety of adverse effects in mammals:1,2 it acts on the central and peripheral nervous system, induces inflammatory response,3 modulates immune functions,3,4 has genetic effects,5 and influences the homeostasis of essential metal ions.6 However, the molecular mechanisms underlying lead toxicity are still not well understood, though it is evident that Pb(II)-nucleic acid interactions may be critical for the toxic action of this metal ion. In fact it is clear that, e.g., Pb(II) ions are efficient in RNA depolymerization.7 Indeed, the Pb(II)* To whom correspondence should be addressed. E-mail: Helmut.Sigel@ unibas.ch. (1) Goyer, R. A. In Handbook on Toxicity of Inorganic Compounds; Seiler, H. G., Sigel, H., Sigel, A., Eds.; Dekker: New York, 1988; pp 359382. (2) Molin Christensen, J.; Kristiansen, J. In Handbook on Metals in Clinical and Analytical Chemistry; Seiler, H. G., Sigel, A., Sigel, H., Eds.; Dekker: New York, 1994; pp 425-440. (3) Ramesh, G. T.; Manna, S. K.; Aggarwal, B. B.; Jadhav, A. L. Toxicol. Appl. Pharmacol. 1999, 155, 280-286. (4) (a) McCabe, M. J., Jr.; Lawrence, D. A. J. Immunol. 1990, 145, 671677. (b) Boscolo, P.; Di Gioacchino, M.; Sabbioni, E.; Reale, M.; Di Giacomo, V.; Giaccio, M.; Giuliano, G. In Metal Ions in Biology and Medicine; Collery, Ph., Bra¨tter, P., Negretti de Bra¨tter, V., Khassanova, L., Etienne, J. C., Eds.; Libbey Eurotext: Paris, 1998; pp 435-439. (5) Johnson, F. M. Mutation Res. 1998, 410, 123-140. (6) (a) Martin, R. B. Met. Ions Biol. Syst. 1986, 20, 21-65. (b) Martin, R. B. In Handbook on Toxicity of Inorganic Compounds; Seiler, H. G., Sigel, H., Sigel, A., Eds.; Dekker: New York, 1988; pp 9-25.

induced hydrolysis of RNA is much studied,8,9 and so-called leadzymes, the activity of which depends on the presence of Pb2+, exist.10-12 Considering that hardly any information exists on the interaction between Pb2+ and nucleotides or related ligands,13-15 we initiated corresponding research and reported recently on the stability of binary complexes formed between Pb2+ and (7) (a) Farkas, W. R. Biochim. Biophys. Acta 1968, 155, 401-409. (b) Werner, C.; Krebs, B.; Keith, G.; Dirheimer, G. Biochim. Biophys. Acta 1976, 432, 161-175. (8) (a) Ohmichi, T.; Sugimoto, N. Biochemistry 1997, 36, 3514-3521. (b) Ciesiolka, J.; Hardt, W.-D.; Schlegl, J.; Erdmann, V. A.; Hartmann, R. K. Eur. J. Biochem. 1994, 219, 49-56. (c) Streicher, B.; von Ahsen, U.; Schroeder, R. Nucleic Acids Res. 1993, 21, 311-317. (9) Brown, R. S.; Dewan, J. C.; Klug, A. Biochemistry 1985, 24, 47854801. (10) (a) Westhof, E.; Hermann, T. Nature Struct. Biol. 1999, 6, 208-209. (b) Wedekind, J. E.; McKay, D. B. Nature Struct. Biol. 1999, 6, 261268. (11) (a) Lemieux, S.; Chartrand, P.; Cedergren, R.; Major, F. RNA 1998, 4, 739-749. (b) Ciesiolka, J.; Michalowski, D.; Wrzesinski, J.; Krajewski, J.; Krzyz˚osiak, W. J. J. Mol. Biol. 1998, 275, 211-220. (12) Katahira, M.; Kim, M. H.; Sugiyama, T.; Nishimura, Y.; Uesugi, S. Eur. J. Biochem. 1998, 255, 727-733. (13) IUPAC Stability Constants Database, Release 3, Version 4.02; compiled by Pettit, L. D., Powell, H. K. J.; Academic Software: Timble, Otley, W. Yorks, UK, 1999. (14) NIST Critically Selected Stability Constants of Metal Complexes, Reference Database 46, Version 5.0; data collected and selected by Smith, R. M., Martell, A. E.; US Department of Commerce, National Institute of Standards and Technology: Gaithersburg, MD, 1998.

10.1021/ic0007207 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/29/2000

5986 Inorganic Chemistry, Vol. 39, No. 26, 2000

Da Costa and Sigel conformation of CMP2- dominates in solution25 and a phosphatecoordinated metal ion thus can not reach the N3/(C2)O site of the cytosine residue. On the other hand, the affinity of Pb2+ is somewhat more pronounced for the cytosine moiety than for the monoprotonated phosphate group as was shown16 for Pb(H;CMP)+ where the proton is at the phosphate group and Pb2+ mainly at the N3/(C2)O site. Therefore, the main question for the present was, Do the purine nucleotides AMP2-, IMP2-, and GMP2- behave like simple phosphate esters or does Pb2+ form a macrochelate with the nucleobase residue? 2. Experimental Section

Figure 1. Chemical structures of adenosine 5′-monophosphate (AMP2-), inosine 5′-monophosphate (IMP2-), and guanosine 5′-monophosphate (GMP2-) in their dominating anti conformation.18,19

simple phosph(on)ate ligands.16 Now we present our results on the stability and structure of the complexes formed between Pb2+ and AMP2-, IMP2-, or GMP2- (Figure 1)17-19 in aqueous solution. The primary binding site for Pb2+, which determines to a very large part the stability of the complexes, is the phosphate group of the nucleotides.16 However, previously it has been shown for several metal ions20,21 such as Mn2+, Cu2+, Zn2+, and Cd2+ that they interact also with N7 of the purine nucleobase residue22 of the nucleotides forming macrochelates20,21 according to equilibrium 1:

Indeed, in several studies dealing with leadzymes10,12 or Pb2+mediated RNA cleavages9,23 as well as for Pb2+-DNA interactions24 evidence is provided that Pb2+ not only interacts with phosphate groups but also with nitrogen and oxygen atoms of nucleobases. Hence, we attempted to quantify in addition the stability of the Pb2+ complexes formed with adenosine (Ado) and guanosine (Guo). Previously we had shown16 that the stability of the Pb(UMP) and Pb(dTMP) complexes is solely determined by the basicity of the phosphate group and that the uracil and thymine moieties are not involved in metal ion binding. This is also true for Pb(CMP),16 since the anti (15) Joint Expert Speciation System (JESS), version 6.0; joint venture by Murray, K., and May, P. M.; Division of Water Technology, CSIR, Pretoria, South Africa, and School of Mathematical and Physical Sciences, Murdoch University, Murdoch, Western Australia, 1999. (16) Da Costa, C. P.; Sigel, H. J. Biol. Inorg. Chem. 1999, 4, 508-514.

2.1. Materials. Adenosine 5′-monophosphoric acid and the disodium salt of guanosine 5′-monophosphate were from Serva Feinbiochemica GmbH, Heidelberg, Germany. The disodium salt of inosine 5′monophosphate, adenosine, and guanosine were from Sigma Chemical Co., St. Louis, MO. The other reagents were the same as used previously.16 All solutions were prepared with deionized, ultrapure (MILLI-Q 185 PLUS, from Millipore S. A., 67120 Molsheim, France), and CO2-free water. The aqueous stock solutions of the ligands were freshly prepared daily, and their exact concentration was newly determined each time by titrations with NaOH. The titer of the NaOH used for the titrations was established with potassium hydrogen phthalate and that of the Pb2+ stock solutions with EDTA. 2.2. Potentiometric pH Titrations. These were carried out with the same equipment and in exactly the same way as described recently.16 In the titrations of adenosine and guanosine, due to the high Pb2+ concentrations needed in the experiments, an interference of Pb2+ with the Cl- ions from the electrolyte of the electrode occurred. To overcome this problem, we used separate electrodes, i.e., a pH measuring electrode (Metrohm 6.0133.100) in combination with an Ag/AgCl reference electrode (Metrohm 6.0726.100) where the outer part was filled with a saturated KNO3 solution. In this way no further interactions were observed and reproducible, high-quality titration curves were obtained. We are grateful to Metrohm AG, Herisau, Switzerland, for providing this information. The determined acidity constants are so-called practical, mixed, or Brønsted constants.26 Their negative logarithms given for aqueous solution at I ) 0.1 M (NaNO3) and 25 °C may be converted into the corresponding concentration constants by subtracting 0.02 from the (17) Abbreviations and definitions. Ado, adenosine; AMP2-, adenosine 5′monophosphate; CMP2-, cytidine 5′-monophosphate; Cyd, cytidine; dTMP2-, thymidine 5′-monophosphate; GMP2-, guanosine 5′-monophosphate; Guo, guanosine; I, ionic strength; IMP2-, inosine 5′monophosphate; Ino, inosine; M2+, general divalent metal ion; NMP2-, nucleoside 5′-monophosphate; Ns, nucleoside; R-PO32-, simple phosphate monoester or phosphonate ligand with R representing a noncoordinating residue (see also legend of Figure 3); UDP3-, uridine 5′-diphosphate; UMP2-, uridine 5′-monophosphate. Species written without a charge either do not carry one or represent the species in general (i.e., independent of their protonation degree); which of the two possibilities applies is always clear from the context. In formulas such as Pb(H;NMP)+, H+ and NMP2- are separated by a semicolon to facilitate reading, yet they appear within the same parentheses to indicate that the proton is at the ligand without defining its location (see sections 3.3-3.5). (18) Aoki, K. Met. Ions Biol. Syst. 1996, 32, 91-134. (19) Tribolet, R.; Sigel, H. Eur. J. Biochem. 1987, 163, 353-363. (20) Sigel, H.; Massoud, S. S.; Corfu`, N. A. J. Am. Chem. Soc. 1994, 116, 2958-2971. (21) (a) Sigel, H.; Song, B. Met. Ions Biol. Syst. 1996, 32, 135-205. (b) Sigel, H. Chem. Soc. ReV. 1993, 22, 255-267. (22) Sigel, H.; Massoud, S. S.; Tribolet, R. J. Am. Chem. Soc. 1988, 110, 6857-6865. (23) (a) Rubin, J. R.; Sundaralingam, M. J. Biomol. Struct. Dyn. 1983, 1, 639-646 (b) Brown, R. S.; Hingerty, B. E.; Dewan, J. C.; Klug, A. Nature 1983, 303, 543-546. (24) SÄ wiatek, J.; Gulanowski, B. Acta Biochim. Pol. 1990, 37, 7-20. (25) Davies, D. B.; Rajani, P.; Sadikot, H. J. Chem. Soc., Perkin Trans. 2 1985, 279-285. (26) Sigel, H.; Zuberbu¨hler, A. D.; Yamauchi, O. Anal. Chim. Acta 1991, 255, 63-72.

Lead(II)-Nucleobase Binding listed pKa values;26 this conversion term contains both the junction potential of the glass electrode and the hydrogen ion activity.26,27 The ionic product of water (Kw) and the conversion term do not enter into our calculation procedures because we evaluate the differences in NaOH consumption between a pair of solutions; i.e., a solution with and one without ligand are always titrated (see also below; for further details refs 26 and 28 may be consulted). The stability constants presented are, as usual, concentration constants. 2.3. Determination of Equilibrium Constants of the Nucleoside Systems. The acidity constants KHH(Guo) and KHGuo of H(Guo)+ and Guo (eqs 2 and 3), respectively, were determined by titrating 25 mL of aqueous 6.6 mM HNO3 (25 °C; I ) 0.1 M, NaNO3) in the presence and absence of 0.93 mM Guo under N2 with 3 mL of 0.06 M NaOH. The constants were calculated with a curve-fitting procedure using a Newton-Gauss nonlinear least-squares program by employing every 0.1 pH unit the difference in NaOH consumption between the two mentioned titrations within the pH range 2.9-10.5 corresponding to about 86% (initial) neutralization for the equilibrium H(Guo)+/Guo and about 95% (final) for Guo/(Guo-H)-. The final results are the averages of six independent pairs of titrations; they were within their error limit identical with the previously measured values.20 2+ complex was The stability constant KPb Pb(Guo) (eq 7) of the Pb(Guo) determined under the conditions given above for the acidity constants, but NaNO3 was partly or fully replaced by Pb(NO3)2 (25 °C; I ) 0.1 M). The ratios Guo:Pb2+ were 1:35.8 and 1:28.7. The experimental data were collected every 0.1 pH unit from the lowest accessible pH (about 2.2) to the beginning of the hydrolysis of Pb(aq)2+ (at pH about 4.4); the latter was evident from the titrations in the absence of ligand. The calculations were done with the same curve-fitting procedure mentioned above for the acidity constants, and in this way an apparent acidity constant pKa′ was obtained, from which the stability constant was calculated as described previously.29 The buffer depression29 was with ∆pKa about 0.2 quite significant. The individual results showed no dependence on the excess of Pb2+ used in the experiments. The final result of the stability constant is the average of 5 independent pairs of titrations. The acidity constant KHH(Ado) (eq 2) was obtained by titrating 50 mL of aqueous 2.4 mM HNO3 in the presence and absence of 0.6 mM Ado with 2 mL of 0.06 M NaOH. Otherwise the conditions were identical with those given above for Guo and also the same evaluation procedure was employed by using the pH range from about 2.8 to 5.6 for the calculations; this range corresponds to a neutralization between about 13% and 99% for the equilibrium H(Ado)+/Ado. The final result is the average of seven independent pairs of titrations and it is within its error limits identical with the value determined previously.19 2+ complex was The stability constant KPb Pb(Ado) (eq 7) of the Pb(Ado) determined under the same conditions used for the acidity constant of H(Ado)+, but part or all of NaNO3 was replaced by Pb(NO3)2 to give Ado:Pb2+ ratios of 1:55.6 and 1:50. The determination of this stability constant was 2-fold hampered: (i) Due to the hydrolysis of Pb(aq)2+ only the pH range from about 3.2 to 4.5 was experimentally accessible. (ii) The buffer depression (see ref 29) ∆pKa equalled only about 0.04 and was thus very small. Consequently, the error in the calculations for KPb Pb(Ado) was very large and the result, which is the average of five independent pairs of titrations, can only be considered as an estimate. 2.4. Determination of the Equilibrium Constants of the Nucleotide Systems. The acidity constants KHH2(GMP) and KHH(GMP) of H2(GMP)( (eqs 4 and 5) were determined by titrating under N2 50 mL of aqueous 3 mM HNO3 (25 °C; I ) 0.1 M, NaNO3) in the presence and absence of 0.6 mM GMP2- (the stock solution was adjusted to pH 8.3) with 2.5 mL of 0.06 M NaOH. The constants were calculated with the above-mentioned curve-fitting procedure in the pH range from 3.2 to 7.0; this means the neutralization degree reached from about 84% to 100% and from 3% to 84% for the equilibria H2(GMP)(/H(GMP)(27) Irving, H. M.; Miles, M. G.; Pettit, L. D. Anal. Chim. Acta 1967, 38, 475-488. (28) Bastian, M.; Sigel, H. J. Coord. Chem. 1991, 23, 137-154. (29) (a) Sigel, R. K. O.; Song, B.; Sigel, H. J. Am. Chem. Soc. 1997, 119, 744-755. (b) Ji, L.-n.; Corfu`, N. A.; Sigel, H. J. Chem. Soc., Dalton Trans. 1991, 1367-1375.

Inorganic Chemistry, Vol. 39, No. 26, 2000 5987 and H(GMP)-/GMP2-, respectively. The results from the seven pairs of titrations made now are within their error limits identical with the previous results.20 Pb + The stability constants KPb(H;GMP) and KPb Pb(GMP) of the Pb(H;GMP) and Pb(GMP) complexes (eqs 10 and 11) were determined under the same conditions, but part of NaNO3 was replaced by Pb(NO3)2 (I ) 0.1 M); the GMP:Pb2+ ratios were 1:27.8, 1:13.9, and 1:6.9. The constants were calculated from the experimentally accessible pH range 2.6-5.0 [beginning of the hydrolysis of Pb(aq)2+] by taking into account the species H+, H2(GMP)(, H(GMP)-, GMP2-, Pb2+, Pb(H;GMP)+, and Pb(GMP). The formation degrees for Pb(H;GMP)+ and Pb(GMP) varied between about 7-40% and 2-30%, respectively. The final results are the averages of seven independent pairs of titrations. The experimental conditions for the titrations with IMP were the same as given above for the GMP systems, but now the acidity constants KHH(IMP) and KHIMP of H(IMP)- were determined (eqs 5 and 6). The pH range from 4.5 to 9.1 was evaluated which corresponds to an initial neutralization degree of 2% for the equilibrium H(IMP)-/IMP2- and of 56% (final) for IMP2-/(IMP-H)3-. The results from six independent pairs of titrations are within the error limits identical with the previous ones.20 The 11 pairs of titrations in the presence of Pb2+ were also made as given above; this also applies to the calculations, but the Pb constant KPb(H;IMP) for the Pb(H;IMP)+ complex could only be estimated. The experimental conditions as well as the evaluations for the determination of the equilibrium constants for the AMP systems ([AMP] ) 0.6 mM) with and without Pb2+ were identical with those used previously16 for the corresponding CMP systems, except that now AMP: Pb2+ ratios of 1:27.8, 1:11, 1:8.3, and 1:5 were used. The evaluations of the eight titration pairs for the acidity constants KHH2(AMP) and KHH(AMP) for H2(AMP)( (eqs 4 and 5) encompassed the pH range 3.37.9 which corresponds to an initial neutralization degree of 23% for the equilibrium H2(AMP)(/H(AMP)- and to a final one of 98% for H(AMP)-/AMP2-; the average results were within their error limits identical with the previous ones.22 The Pb2+/AMP titrations could only be evaluated in the pH range 3.2-4.6, which corresponds to formation degrees of about 1-9.5% for Pb(H;AMP)+ and 0.5-7.5% for Pb(AMP) because of the formation of a precipitate which clearly occurred before the onset of the hydrolysis of Pb(aq)2+. The final results for the stability Pb constants, KPb(H;AMP) and KPb Pb(AMP) (eqs 10 and 11), are the averages of 10 pairs of titrations.

3. Results and Discussion All potentiometric pH titrations (I ) 0.1 M, NaNO3; 25 °C) were carried out at ligand concentrations below 1 mM, usually at 0.6 mM. Under these conditions self-stacking of the nucleosides and the NMPs of Figure 1 is negligible.20 Hence, all the results presented below apply to monomeric species. 3.1. Acidity Constants of the Protonated Nucleosides and Nucleotides. The nucleosides (Ns) adenosine (Ado) and guanosine (Guo) can both accept a proton at the purine ring, Ado at N1 and Guo at N7.20 Consequently, the following equilibrium needs to be considered for both nucleosides:

H(Ns)+ h Ns + H+ KHH(Ns)

+

(2a) +

) [Ns][H ]/[H(Ns) ]

(2b)

The neutral guanosine may loose a further proton from the (N1)H site, giving rise to equilibrium 3a:

Guo h (Guo-H)- + H+ KHGuo

-

+

) [(Guo-H) ][H ]/[Guo]

(3a) (3b)

Clearly, the nucleoside 5′-monophosphates (NMP2-) shown in Figure 1 can bind three protons, two at the phosphate group and one at the purine moiety. The first proton is released from

5988 Inorganic Chemistry, Vol. 39, No. 26, 2000

Da Costa and Sigel

Table 1. Negative Logarithms of the Acidity Constants (Eqs 2-6)]a of the Monoprotonated Nucleosides, H(Ns)+, and the Twofold Protonated Nucleoside 5′-Monophosphates, H2(NMP)(, Considered in This Study and as Determined by Potentiometric pH Titrations in Aqueous Solution at 25 °C and I ) 0.1 M (NaNO3)b,c pKHH(Ns)

acid

or pKHH2(NMP) (eqs 2, 4)

H(Ado)+ H(Ino)+ H(Guo)+ H2(AMP)( H2(IMP)( H2(GMP)(

3.61 ( 0.03d (1.06 ( 0.06)20,30 2.11 ( 0.04 3.84 ( 0.02d (1.30 ( 0.10)20 2.48 ( 0.04

pKHH(NMP) (eq 5)

pKHNs

or pKHNMP (eqs 3, 6)

6.21 ( 0.01 6.22 ( 0.01 6.25 ( 0.02

(8.76 ( 0.03)20 9.22 ( 0.02e 9.02 ( 0.02 (9.49 ( 0.02)20

a So-called practical (or mixed) acidity constants26 are listed; see section 2.2. b The error limits are 3 times the standard error of the mean value (3σ) or the sum of the probable systematic errors, whichever is larger. c The values in parentheses are given for comparison; they are taken from the indicated references. All the other acidity constants have been redetermined during this work (see sections 2.3 and 2.4); the results were identical within their error limits with the published values which therefore are again listed above; they are taken from refs 19 (Ado), 20 (Guo, IMP, GMP) and 22 (AMP). d These values refer to the deprotonation of the (N1)H+ site of the adenine residue; all the other values in this column refer to the deprotonation of a (N7)H+ site. e This error limit was enlarged from (0.01 in ref 20 to (0.02 to be in accord with the experiments carried out now.

the -P(O)(OH)2 group at a rather low pH (pKa e 0.5)20 and is not of relevance for the present study. The second proton (eq 4) is released from the (N1)H+ site of H2(AMP)( or the (N7)H+ site of H2(IMP)( and H2(GMP)( followed by the proton from the -P(O)2(OH)- group (eq 5). Finally, IMP2- and GMP2may be deprotonated at their (N1)H site leading to the (NMPH)3- species (eq 6). Hence, the following three equilibria are relevant for this work:

H2(NMP)( h H(NMP)- + H+

(4a)

KHH2(NMP) ) [H(NMP)-][H+]/[H2(NMP)(]

(4b)

H(NMP)- h NMP2- + H+

(5a)

H KH(NMP)

+

-

) [NMP ][H ]/[H(NMP) ] 2-

(5b)

NMP2- h (NMP-H)3- + H+

(6a)

KHNMP ) [(NMP-H)3-][H+]/[NMP2-]

(6b)

We have measured by potentiometric pH titrations the pertinent acidity constants which all agreed within their error limits with the values published earlier by our group.19-22,26,30 The corresponding results are listed in Table 1, and the site attributions of the protons as described above are confirmed by these data. 3.2. Stability Constants of Pb2+ Complexes Formed with Nucleosides. Various metal ions (such as Cu2+, Ni2+) can form complexes with adenosine31,32 and guanosine.33 Therefore, we also attempted to measure stability constants of the corresponding Pb(Ns)2+ complexes. Since the stability of these complexes is expected to be low, a large excess of Pb2+, compared to Ns, was used in the experiments (see section 2.3). Indeed, the experimental data could be fully explained by taking into (30) Corfu`, N. A.; Sigel, H. Eur. J. Biochem. 1991, 199, 659-669. (31) Sigel, H.; Corfu`, N. A.; Ji, L.-n.; Martin, R. B. Comments Inorg. Chem. 1992, 13, 35-59. (32) Martin, R. B. Met. Ions Biol. Syst. 1996, 32, 61-89. (33) Song, B.; Zhao, J.; Griesser, R.; Meiser, C.; Sigel, H.; Lippert, B. Chem. Eur. J. 1999, 5, 2374-2387.

account equilibria 2a and 7a as long as the evaluation of the

Pb2+ + Ns h Pb(Ns)2+

(7a)

Pb KPb(Ns) ) [Pb(Ns)2+]/([Pb2+][Ns])

(7b)

data was not carried into the pH range where hydrolysis of Pb(aq)2+ occurs (see section 2.3). The stability constants given below for the Pb(Ado)2+ and Pb(Guo)2+ complexes (eq 7) are the average of five independent experiments for each system; they refer to aqueous solutions at 25 °C and I ) 0.1 M (for the error limits see footnote b in Table 1):

log KPb Pb(Ado) ) 0.4 ( 0.3

(8)

log KPb Pb(Guo) ) 1.25 ( 0.17

(9)

Pb The determination of KPb(Guo) (eq 9) was straightforward, Pb whereas KPb(Ado) (eq 8) is only an estimate because the measurements were hampered not only by the hydrolysis of Pb(aq)2+, but also by the instability of Pb(Ado)2+ which leads to a very low buffer depression (∆pKa) between titrations of the Ado and the Ado/Pb2+ systems (see section 2.3). Indeed, in an early attempt to measure this stability constant, it was concluded34 that the association of Pb2+ with Ado is negligible. In the same study34 also a constant for Pb(Guo)2+ was Pb ) 0.48 ( 0.14 (20 °C; I ) 1 M, measured: log KPb(Guo) NaNO3). Though this value is considerably smaller than the present result (eq 9), the formation of the Pb(Guo)2+ complex is confirmed; aside from possible difficulties due to the way the earlier experiments were performed,34 the discrepancy between the constants probably stems from the use of a 10 times higher ionic strength (background electrolyte) in the earlier study. In M(Guo)2+ complexes the metal ions are coordinated to N7,33 which is the most basic site, though possibly20 also semichelates form involving a hydrogen bond between a M2+coordinated H2O molecule and the carbonyl oxygen at C6. For M(Ado)2+ complexes it was previously shown that a N1 versus N7 dichotomy occurs,31,32 N7 binding being favored for most metal ions, probably also for Pb2+ (see also the considerations in section 3.7). Finally, it needs to be added that the stability of Pb(cytidine)2+, where Pb2+ coordinates via the N3/(C2)O site, Pb ) 1.20 ( 0.07,16 is very similar to the one of with log KPb(Cyd) 2+ Pb(Guo) (see eq 9). 3.3. Stability Constants of Pb2+-Nucleotide Complexes. The experimental data of the potentiometric pH titrations (see section 2.4) of the three Pb2+/NMP systems, where NMP ) AMP, IMP, or GMP, allow the determination of the stability constants defined by equilibria 10a and 11a:

Pb2+ + H(NMP)- h Pb(H;NMP)+ Pb KPb(H;NMP)

+

-

(10a)

) [Pb(H;NMP) ]/([Pb ][H(NMP) ])

(10b)

Pb2+ + NMP2- h Pb(NMP)

(11a)

Pb KPb(NMP)

2+

) [Pb(NMP)]/([Pb ][NMP ]) 2+

2-

(11b)

Overall, equilibria 4a, 5a, 10a, and 11a are sufficient to obtain excellent fitting of the titration data, provided the evaluation is not carried into the pH range where hydrolysis of Pb(aq)2+ occurs, which was evident from the titrations without ligand. (34) Fiskin, A. M.; Beer, M. Biochemistry 1965, 4, 1289-1294.

Lead(II)-Nucleobase Binding

Inorganic Chemistry, Vol. 39, No. 26, 2000 5989

Table 2. Logarithms of the Stability Constants of the Pb(H;NMP)+ (Eq 10) and Pb(NMP) Complexes (Eq 11), Together with the Negative Logarithms of the Acidity Constants for the Pb(H;NMP)+ Species (Eqs 12 and 13), as Determined by Potentiometric pH Titrations in Aqueous Solution at 25 °C and I ) 0.1 M (NaNO3)a NMP2-

Pb log KPb(H;NMP)

log KPb Pb(NMP)

H pKPb(H;NMP)

AMP2IMP2GMP2-

1.08 ( 0.04 1.30 ( 0.15b 1.52 ( 0.10

2.92 ( 0.08 3.06 ( 0.05 3.23 ( 0.08

4.37 ( 0.09 4.46 ( 0.16 4.54 ( 0.13

a

See footnote b in Table 1; the error limits of the derived data, in H the present case for pKPb(H;NMP) , were calculated according to the error propagation after Gauss. b This value is an estimation which is mainly based on the lower basicity of N7 in the hypoxanthine moiety compared to that in the guanine residue (cf. Table 1).

The pH range where the N1-deprotonated Pb(IMP-H)- or Pb(GMP-H)- species might be formed is not reached. Of course, equilibria 10a and 11a are also connected via equilibrium 12a and the corresponding acidity constant (eq 12b) may be calculated with eq 13:

Pb(H;NMP)+ h Pb(NMP) + H+ H KPb(H;NMP)

+

(12a) +

) [Pb(NMP)][H ]/[Pb(H;NMP) ] (12b)

H H Pb Pb ) pKH(NMP) + log KPb(H;NMP) - log KPb(NMP) pKPb(H;NMP)

(13) The results are listed in Table 2. None of these values has been determined before.13-15 Application of the constants of Tables 1 and 2 allows calculation of the formation degree of the various species as a function of pH. Two representative examples are shown in Figure 2; the concentrations used are close to some employed in the experiments. Since the acidity constants of the H(NMP)- species are nearly identical (Table 1, column 3), an important conclusion follows immediately from the stability constants listed in column 3 of Table 2 for the Pb(NMP) complexes: There is a stability increase in the series, Pb(AMP) < Pb(IMP) < Pb(GMP), and this is only possible if the phosphate-coordinated Pb2+ interacts to some extent also with N7 of the nucleobases; hence, equilibrium 1 exists. The increased stability of Pb(GMP), compared to that of Pb(AMP), is also evident from Figure 2. A quantitative evaluation of this observation is given in section 3.8. 3.4. Structural Considerations on the Monoprotonated Pb(H;NMP)+ Complexes. The Proton is at the Phosphate Group! Potentiometric pH titrations allow determination of the stability constants of Pb(H;NMP)+ complexes, but in order to locate the binding sites of the proton and the metal ion in these species, further information is needed. At first one best considers the proton because binding of a metal ion to a protonated ligand commonly leads to an acidification of the ligand-bound proton.33,35 Indeed, the acidity constants of the Pb(H;NMP)+ H complexes given in column 4 of Table 2 (pKPb(H;NMP) = 4.45) are on average 1.8 pK units smaller than the values listed in H column 3 of Table 1 for the H(NMP)- species (pKH(NMP) = + 6.23), but the acidity constants of the Pb(H;NMP) complexes are also between about 0.6 and 3.2 pK units larger than the pKHH2(NMP) values (see column 2 in Table 1); hence, the proton must be located at the phosphate group of the NMPs in the Pb(H;NMP)+ complexes. (35) Sigel, H.; Lippert, B. Pure Appl. Chem. 1998, 70, 845-854.

Figure 2. Effect of pH on the concentration of species present in the Pb2+ systems with GMP (top) and AMP (bottom); the results are given as percentages of the total NMP concentration present. They were computed with the determined acidity (Table 1) and stability constants (Table 2) by using [NMP]tot ) 0.0006 M and [Pb2+]tot ) 0.006 M (concentrations close to the experimental conditions; see section 2.4). In the pH range above 5 hydroxo complex formation occurs (see section 2.4), and this was ignored in the above plots.

However, where is the metal ion? Tentatively one might argue that if the proton is at the phosphate group then it appears likely that Pb2+ is at the nucleobase residue. In fact, the increasing stability of the Pb(H;NMP)+ complexes in the order Pb(H;AMP)+ < Pb(H;IMP)+ < Pb(H;GMP)+ supports this suggestion because the binding tendency of Pb2+ toward the -P(O)2(OH)- residue should be identical for all three nucleotides, since this residue has the same basicity (Table 1, column 3). These tentative reasonings are largely confirmed by the following evaluation. 3.5. Considerations on the Location of Pb2+ in the Pb(H;NMP)+ Complexes. For the location of Pb2+, in principle two possibilities exist: (i) the metal ion is at the phosphate group like the proton, symbolized by (NMP‚Pb‚H)+; and (ii) it is at the nucleobase, symbolized by (Pb‚NMP‚H)+. Hence, eq 10b may be rewritten in the form of eq 14: Pb KPb(H;NMP) )

[(Pb‚NMP‚H)+] + [(NMP‚Pb‚H)+] [Pb2+][H(NMP)-]

Pb ) kPb Pb‚NMP‚H + kNMP‚Pb‚H

(14a) (14b)

Pb Estimations for the micro stability constant kPb‚NMP‚H may be made by using the known stability constants of the Pb(Ns)2+ complexes (section 3.2). Pb ) For the GMP system, the stability constant log KPb(Guo) 2+ 1.25 ( 0.17 (see eq 9) of Pb(Guo) needs to be corrected (i)

5990 Inorganic Chemistry, Vol. 39, No. 26, 2000 for the different basicities of the N7 site in H(GMP)- and Guo and (ii) for the charge effect that the -P(O)2(OH)- group exerts on the Pb2+ bound at N7 of the guanine residue in (Pb‚GMP‚ Pb ) 1.76 ( 0.23 H)+. This estimation36 results in log kPb‚GMP‚H and this value is evidently identical within the error limits with Pb the measured value, log KPb(H;GMP) ) 1.52 ( 0.10, meaning that the stability of the Pb(H;GMP)+ species is determined by the stability of the (Pb‚GMP‚H)+ isomer (cf. eq 14) which carries Pb2+ at N7 and the proton at the phosphate group and that the formation of the (GMP‚Pb‚H)+ isomer with both Pb2+ and H+ at the phosphate group is negligible. The same procedure may be applied for the AMP system by Pb Pb ) 0.4 ( 0.3 (eq 8). This gives37 log kPb‚AMP‚H using log KPb(Ado) ) 0.90 ( 0.35 for the stability constant of the (Pb‚AMP‚H)+ isomer. This micro stability constant is again within its error Pb ) 1.08 limits identical with the measured value log KPb(H;AMP) ( 0.04. Hence, it seems that also in this case the (Pb‚AMP‚ H)+ isomer with Pb2+ at the adenine residue and H+ at the phosphate group dominates. However, at this point one may also ask, How stable is the (NMP‚Pb‚H)+ isomer? Evidently, the stability of this isomer should only depend on the affinity of the -P(O)2(OH)- group for Pb2+, and therefore it should be the same for the AMP, IMP, and GMP systems. Unfortunately, no such value is available in the literature.13-15 The stability of the Pb(H2PO4)+ complex, Pb ) 1.5 ( 0.5,38 appears to be too large to be used log KPb(H 2PO4) here because comparisons of the stabilities of M(HPO4) complexes with those of the M(CH3OPO3) species show39 that the latter complexes are somewhat less stable. Based on the stability constants of diphosphate monoesters we estimate40,41 Pb log kNMP‚Pb‚H ) 0.7 ( 0.4 for the stability of the (NMP‚Pb‚H)+ isomer which carries Pb2+ and H+ at the phosphate group. Pb Comparison of this value with log kPb‚AMP‚H ) 0.9 ( 0.35 reveals that both microconstants are of the same order; in fact, Pb ) log [10(0.9(0.35) + application of eq 14b gives log KPb(H;AMP) (0.7 ( 0.4) 10 ] ) 1.1 ((0.3). Evidently, this calculated value also Pb (36) The stability constant of Pb(Guo)2+, log KPb(Guo) ) 1.25 ( 0.17 (eq 9), is corrected for the different basicities of N7 in H(GMP)- and H Guo [i.e., ∆ pKa ) pKHH2(GMP) - pKH(Guo) ) (2.48 ( 0.04) - (2.11 ( 0.04) ) 0.37 ( 0.06] by applying the estimated slope m ) 0.3 for the regression line of the log K versus pKa plot (this estimate is based on the slopes given in refs 31 and 32) and this leads to the “corrected” value (1.25 ( 0.17) + (0.11 ( 0.06/estimated error) ) 1.36 ( 0.18. This value needs to be further corrected for the charge effect which the -P(O)2(OH)- group exerts on Pb2+ at N7 [the effect of the same group on (N7)H+ is taken care of via ∆pKa]; this effect corresponds to 0.40 ( 0.15 log unit, as is known28 from various other cases where the distances between the positive and negative charges are of a Pb comparable size. Hence, one obtains log kPb‚GMP‚H ) (1.36 ( 0.18) + (0.40 ( 0.15) ) 1.76 ( 0.23. (37) Application of the procedure described in footnote 36 gives the correction term [(3.84 ( 0.02) - (3.61 ( 0.03) ) 0.23 ( 0.04 and multiplied by m ) 0.3] 0.07 ( 0.04 for the different basicities of N1 in AMP2- and Ado; this together with the charge effect of 0.40 ( Pb ) (0.4 ( 0.3) 0.15 leads to the micro stability constant log kPb‚AMP‚H + (0.07 ( 0.04) + (0.40 ( 0.15) ) 0.9 ( 0.35. (38) Nriagu, J. O. Inorg. Chem. 1972, 11, 2499-2503. (39) Saha, A.; Saha, N.; Ji, L.-n.; Zhao, J.; Grega´nˇ, F.; Sajadi, S. A. A.; Song, B.; Sigel, H. J. Biol. Inorg. Chem. 1996, 1, 231-238. (40) Protonation of the β-phosphate group in M(UDP)- complexes (where M2+ ) Cu2+, Zn2+, Cd2+) destabilizes these complexes by about 2.1 log units.41 Since in these cases M2+ coordinates to the R- and β-phosphate groups, the effect of the proton is expected to be somewhat larger in the (NMP‚Pb‚H)+ complexes, where only a single Pb ) phosphate group is available. We estimate therefore log kNMP‚Pb‚H 2.9-2.2 ) 0.7 ( 0.4 (estimated error/the value 2.9 is from column 3 in Table 2). (41) Sajadi, S. A. A.; Song, B.; Grega´nˇ, F.; Sigel, H. Inorg. Chem. 1999, 38, 439-448.

Da Costa and Sigel Pb ) 1.08 ( 0.04 agrees with the measured one, log KPb(H;AMP) (Table 2). Application of the two estimated micro stability constants37,40 allows calculation of the ratio R for the two isomers:

R) )

[(Pb‚AMP‚H)+] [(AMP‚Pb‚H)+] 10(0.9(0.35) 10

(0.7(0.4)

)

)

Pb kPb‚AMP‚H Pb kAMP‚Pb‚H

1.6 60 = 1 40

(15a)

(15b)

Of course, the result of eq 15 must be considered as a rough estimate, but it indicates that most probably both isomers, (Pb‚ AMP‚H)+ and (AMP‚Pb‚H)+, occur in aqueous solution with a possible dominance of the isomer with Pb2+ at the adenine residue and the proton at the phosphate group. On the other hand, the above reasonings also indicate that the affinity of the adenine residue and that of a -P(O)2(OH)- group for Pb2+ are of comparable size. Application of eq 14b to the GMP system gives log Pb KPb(H;GMP) ) log [10(1.76(0.23) + 10(0.7(0.4)] ) 1.8 ((0.3) which is still within its error limits identical with the measured one, Pb log KPb(H;GMP) ) 1.52 ( 0.10 (Table 2). Calculation of the ratio R ) [(Pb‚GMP‚H)+]/[(GMP‚Pb‚H)+] ) 10(1.76(0.23)/10(0.7(0.4) ) 11.5/1 ) 92/8 confirms the above conclusion that the isomer (Pb‚GMP‚H)+, with Pb2+ at N7 and H+ at the phosphate group, strongly dominates with a formation degree of over 90%. An analogous evaluation is not possible for Pb(H;IMP)+ because the stability of the Pb(Ino)2+ complex is unknown, but based on the results obtained for Pb(H;AMP)+ and especially for the more closely related Pb(H;GMP)+ system, it is safe to suggest that in this system also the (Pb‚IMP‚H)+ isomer with Pb2+ at N7 and H+ at the phosphate group dominates. To conclude, the various evaluations in this section show that Pb2+ clearly has an affinity for the nucleobase residues considered here (Table 2); this affinity decreases in the order guanine > hypoxanthine > adenine in agreement with the results described in section 3.2. 3.6. Evaluation of the Stability of the Pb(NMP) Complexes. We have already noted in section 3.3 the increase in stability in the series Pb(AMP) < Pb(IMP) < Pb(GMP). Indeed, any macrochelate formation as described by equilibrium 1 must be reflected in an enhanced complex stability.20,22,42 Therefore, the stability of the Pb(NMP) complexes is now evaluated by making use of the previously established16 straightPb H versus pKH(R-PO plot, line correlation for a log KPb(R-PO 3) 3) 2where R-PO3 represents phosphate monoester or phosphonate ligands16,43 in which the residue R is unable to interact with the metal ion (eq 16):16 Pb H ) (m)pKH(R-PO +b log KPb(R-PO 3) 3)

(16a)

H ) (0.493 ( 0.033)pKH(R-PO 3)

(0.122 ( 0.213) (16b) The error limits of log stability constants calculated with given H pKH(R-PO values and eq 16 are (0.08 log unit (3σ) in the pKa 3) range 5-8.16 This means that with a known pKa value for the deprotonation of a -P(O)2(OH)- group an expected stability constant for a phosph(on)ate-Pb2+ complex can be calculated. (42) Martin, R. B.; Sigel, H. Comments Inorg. Chem. 1988, 6, 285-314. (43) Sigel, H.; Chen, D.; Corfu`, N. A.; Grega´nˇ, F.; Holy´, A.; Strasˇa´k, M. HelV. Chim. Acta 1992, 75, 2634-2656.

Lead(II)-Nucleobase Binding

Inorganic Chemistry, Vol. 39, No. 26, 2000 5991 Table 3. Stability Constant Comparison for the Pb(NMP) Complexes between the Measured Stability Constants (exptl; Table 2, Column 3) and the Calculated Stability Constants (calcd) Based on the Basicity of the Phosphate Group in AMP2-, IMP2-, and GMP2- (pKHH(NMP); Table 1, Column 3) and the Baseline Equation Established Previously,16 (see Eq 16 and Figure 3), Together with the Stability Differences log ∆Pb/NMP as Defined by Eq 17 (Aqueous Solution; 25 °C; I ) 0.1 M, NaNO3)a NMP22-

AMP IMP2GMP2a

Figure 3. Evidence for an enhanced stability of the Pb2+ 1:1 complexes formed with IMP2- and GMP2-, and for the lack of such an enhanced stability of the Pb(AMP) complex (b), based on the relationship Pb H between log KPb(R-PO and pKH(R-PO for the 1:1 complexes of Pb2+ 3) 3) with some simple phosphate monoester or phosphonate ligands (RPO32-) (O): 4-nitrophenyl phosphate (NPhP2-), phenyl phosphate (PhP2-), uridine 5′-monophosphate (UMP2-), D-ribose 5-monophosphate (RibMP2-), thymidine [) 1-(2′-deoxy- β-D-ribofuranosyl)thymine] 5′monophosphate (dTMP2-), n-butyl phosphate (BuP2-), methanephosphonate (MeP2-), and ethanephosphonate (EtP2-) (from left to right). The least-squares line (eq 16) is drawn through the corresponding eight data sets (O), which are taken from ref 16. The data points due to the equilibrium constants for the Pb2+/AMP, /IMP, and /GMP systems (b) are based on the data given in columns 3 of Tables 1 and 2. The vertical broken lines emphasize the stability differences of the Pb(NMP) (b) complexes to the reference line; these differences are equal to log ∆Pb/NMP (eq 17), the values of which are listed in column 4 of Table 3. All the plotted equilibrium constant values refer to aqueous solutions at 25 °C and I ) 0.1 M (NaNO3). Pb H The plot of log KPb(R-PO versus pKH(R-PO according to eq 3) 3) 16 is shown in Figure 3 for the 1:1 complexes of Pb2+ with eight simple ligands allowing only a phosph(on)ate-Pb2+ coordination. The solid points referring to the Pb2+ complexes of AMP2-, IMP2-, and GMP2- prove an increased stability for the IMP and GMP systems. This observation can be evaluated quantitatively by calculating with the straight-line eq 16 and H the pKH(NMP) values the expected (calcd) stabilities for the Pb(NMP) complexes having solely a phosphate-Pb2+ coordination. The corresponding results are listed in column 3 of Table 3. Comparison of the calculated (calcd) stability constants for the Pb(NMP) complexes with the measured (exptl) ones, i.e., according to eq 17a, leads to the stability differences given in

Pb Pb log ∆Pb/NMP ) log KPb(NMP) - log KPb(NMP) exptl calcd Pb Pb ) log KPb(NMP) - log KPb(NMP) op

(17a) (17b)

Pb the fourth column of Table 3. The expression KPb(NMP) (eq exptl Pb 17a) corresponds of course to KPb(NMP) as used in Table 2 and Pb Pb and KPb(NMP) are synonymous (eq 17b) because KPb(NMP) op calcd the calculated value equals the stability constant of the “open” isomer, Pb(NMP)op, of equilibrium 1 in which only a -PO32-/ Pb2+ interaction occurs (see also section 3.8).

Pb log KPb(NMP)exptl

Pb log KPb(NMP)calcd

log ∆Pb/NMP

2.92 ( 0.08 3.06 ( 0.05 3.23 ( 0.08

2.94 ( 0.08 2.94 ( 0.08 2.96 ( 0.08

-0.02 ( 0.11 0.12 ( 0.09 0.27 ( 0.11

See footnote a in Table 2.

3.7. Macrochelate Formation and Properties of the N7 Sites. The vertical broken lines seen in Figure 3 correspond to the log ∆Pb/NMP values (eq 17) given in column 4 of Table 3, and these prove for the Pb(IMP) and Pb(GMP) complexes an increased stability whereas the one of the Pb(AMP) complex is determined within the error limits by the basicity of the phosphate group. Previously it has been proven for several metal ions, including Zn2+ and Cd2+, by using tubercidine 5′monophosphate (TuMP2- ) 7-deaza-AMP2-), that in the M(AMP) complexes macrochelate formation, which is responsible for the increased stability, occurs with N7.22 Consequently, one expects that the extent of macrochelate formation depends on the basicity of N7, and indeed, this has also been proven for several series of M(AMP), M(IMP), and M(GMP) complexes.20 In the present situation the basicity of N7 is best described H values given for H(Ino)+ and in a relatiVe sense by the pKH(Ns) + H(Guo) in Table 1, since the effect of the -P(O)2(OH)residue on N7 basicity is the same for all three NMPs considered here. However, for adenosine the situation is more complicated because the initial protonation of the purine ring occurs at N1 and not at N7; hence, a micro acidity constant quantifying the N7 basicity has to be determined in an indirect way and over the years several attempts have been made.31,32,44,45 The micro acidity constant needed now is a relatiVe one, i.e., one that takes the steric inhibition46 exercized by the (C6)NH2 group on metal H ion coordination at the N7 site into account, i.e., pKH(N7/Ado,NH 2) 31,47,48 ) -0.2 ( 0.3. In inosine and guanosine no competition between the proton affinity of N7 and another site exists; i.e., the measured macroconstants are identical with the correspondH H ing microconstants, pKH(Ns) ) pkH(N7/Ns) (see Table 1). Since the log ∆ values according to eq 17 reflect the extent of macrochelate formation, they are plotted in Figure 4 as a H , discussed function of the micro acidity constants, pkH(N7/Ns) above; the result for the three Pb(NMP) complexes is a straight line. In addition, the log ∆M/NMP values for the Zn(NMP) and Cd(NMP) complexes21a are plotted in the same figure to demonstrate that Pb2+ is not a special case but behaves in its NMP2- complexes just as other metal ions with an affinity for N7. Only the affinity of Pb2+ toward N7 is somewhat less pronounced (see Figure 4) than that of Zn2+ or Cd2+.20,21 The straight lines seen in Figure 4 do not exclude the possibility (44) Martin, R. B. Acc. Chem. Res. 1985, 18, 32-38. (45) Kinjo, Y.; Tribolet, R.; Corfu`, N. A.; Sigel, H. Inorg. Chem. 1989, 28, 1480-1489. (46) Kapinos, L. E.; Song, B.; Holy´, A.; Sigel, H. Chimia 1996, 50, 334 (No. 121). (47) See also ref 21a, section 4.4 on p 167. (48) The simple micro acidity constant for protonation at the N7 site of adenosine (i.e., without the consideration of the steric effect of the (C6)NH2 group on metal ion binding at N7) has been estimated H ) 1.3.32 recently by Martin: pkH(N7/Ado)

5992 Inorganic Chemistry, Vol. 39, No. 26, 2000

Da Costa and Sigel Table 4. Extent of Macrochelate Formation According to Equilibrium 1 for Pb(NMP) Complexes as Calculated from the Stability-Constant Differences, log ∆Pb/NMP (Eq 17; Table 3, Column 4): Given Are the Intramolecular and Dimensionless Equilibrium Constant KI (Eqs 18 and 19) and Percentage (Eq 20) of the Closed Isomer, Pb(NMP)cl, in Aqueous Solution at 25 °C and I ) 0.1 M (NaNO3)a system

log ∆Pb/NMP

KI

% Pb(NMP)cl

Pb(AMP) Pb(IMP) Pb(GMP)

-0.02 ( 0.11 0.12 ( 0.09 0.27 ( 0.11

0 ( adenine (0.8) > uracil = thymine. The log affinity constants given in parentheses in the above series are based on the measured stability constants of the Pb(Ns)2+ (section 3.2) and Pb(H;NMP)+ (Table 2) complexes as well as on the estimated stabilities for the (Pb‚NMP‚H)+ species (section 3.5). These constants reflect the affinities for Pb2+ of the N7[(C6)O], N3/(C2)O, and N7/(N1) sites of the guanine, cytosine, and adenine residues, respectively, in single-stranded nucleic acids. The affinities of the adenine residue (log KPb = 0.8) and the phosphodiester linkage, -O-P(O)2--O- (∼0.7), are very similar, but the latter is overall more likely to coordinate with Pb2+ because of its higher abundance. The (C2)O and (C4)O carbonyl sites of uracil and thymine are expected to interact only weakly with Pb2+. To summarize, the primary binding sites for Pb2+ in a nucleic acid are the guanine and cytosine residues, followed by the -OP(O)2--O- unit due to its abundance, whereas N7 and N1 of adenine and especially the carbonyl oxygens of uracil and thymine are expected to interact with Pb2+ only if they are preoriented in a sterically favorable position by the initial or primary metal ion binding site.49a The latter also applies to the hydroxy groups of ribose residues: such -OH groups are known49b,c to be able to interact with metal ions, but only weakly.

Lead(II)-Nucleobase Binding

Inorganic Chemistry, Vol. 39, No. 26, 2000 5993

With the above conclusions in mind it is interesting to note that in leadzyme, a ribozyme that requires Pb2+, the cleavage site of the RNA is between a guanine and cytosine residue,8a,10a,11a though this does not necessarily mean that the metal ion is bound here. However, there is now much evidence11a,12,50,51 including an X-ray structure10b that N7 [and possibly also (C6)O] of a guanine residue is essential for Pb2+ binding and activity. Indeed, alterations at several of the guanine residues in leadzyme prove that these sites are crucial for the catalytic activity;11a apparently only three nucleobases, one cytosine and two guanines, are absolutely required for catalysis.51 On the other hand, in the X-ray structure study,10b aside from -O-P(O)2-O-/Pb2+ binding, also a N1(adenine)-Pb2+ interaction is proposed, but this residue can be replaced by several other

residues without severely impairing activity.10a,50 In another leadzyme, which has two sites for Pb2+ binding, also guanine residues are essential.12 It has further been suggested that a leadbound hydroxide ion in Pb(OH)+ serves to activate the 2′hydroxyl group of the cytidine residue.10b,52 This is similar to the M(OH)+-facilitated hydrolysis of nucleoside 5′-triphosphates where evidence exists53 that an intramolecular nucleophilic attack of the coordinated hydroxide ion occurs (see also ref 54).

(49) (a) For further and more detailed information, see: Sigel, H.; Da Costa, C. P.; Martin, R. B. Coord. Chem. ReV. 2001, in press. (b) Liang, G.; Chen, D.; Bastian, M.; Sigel, H. J. Am. Chem. Soc. 1992, 114, 77807785. (c) Sigel, H.; Kapinos, L. E. Coord. Chem. ReV. 2000, 200202, 563-594. (50) Chartrand, P.; Usman, N.; Cedergren, R. Biochemistry 1997, 36, 31453150. (51) Hoogstraten, C. G.; Legault, P.; Pardi, A. J. Mol. Biol. 1998, 284, 337-350.

IC0007207

Acknowledgment. The competent technical assistance of Mrs. Rita Baumbusch in the preparation of the manuscript and research grants from the Swiss National Science Foundation and the Swiss Federal Office for Education & Science (COST D8) are gratefully acknowledged.

(52) Pan, T.; Dichtl, B.; Uhlenbeck, O. C. Biochemistry 1994, 33, 95619565. (53) (a) Sigel, H. Coord. Chem. ReV. 1990, 100, 453-539. (b) Sigel, H. Inorg. Chim. Acta 1992, 198-200, 1-11. (c) Sigel, H. Pure Appl. Chem. 1998, 70, 969-976. (54) Sigel, H. Pure Appl. Chem. 1999, 71, 1727-1740.